Method of quickly producing antibodies against avian influenza and maintain antibody titer of duck

ABSTRACT

The present invention provides a method for enabling ducklings to quickly produce anti-avian influenza antibodies and maintain antibody titer, including: step 1) the first immunization is performed on ducklings at the age of 5 to 15 days: each duckling is inoculated with an avian influenza inactivated antigen in an abdomen, and simultaneously intramuscularly or subcutaneously immunized with an avian influenza inactivated oil-emulsion vaccine. In the method, through the double effects of intraperitoneal inoculation with the inactivated antigen and intramuscular or subcutaneous injection with the inactivated oil-emulsion vaccine on the ducklings, the ducklings can quickly produce an immune response so as to quickly produce the anti-avian influenza antibodies; and the immune dead time of the ducklings immunized with the avian influenza inactivated vaccine can be effectively reduced by more than 7 days.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, Chinese Patent Application No.201610105195.5 with a filing date of Feb. 25, 2016. The content of theaforementioned application, including any intervening amendmentsthereto, is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to the technical field of waterfowlbreeding, and in particular relates to a method for enabling ducklingsto quickly produce anti-avian influenza antibodies and maintain antibodytiter.

BACKGROUND

Since the end of the last century, the virulence of a high-pathogenicityH5 subtype epidemic strain to the waterfowl has been graduallyincreased, and also has led to the disease and death of the waterfowllike the terrestrial poultry. The waterfowl itself is not only a libraryof influenza viruses, but also susceptible host of influenza viruses.

An acquired immune system of the fowl consists of bursal derivedimmunity and thymus derived immunity. Antibodies are produced throughthe stimulation of antigens, and three types of antibodies are producedthrough the cellular immune response: IgM, IgY and IgA. Li Ning teamfinds that the number of immune-related genes of ducklings is equal tothat of immune-related genes of chickens, both obviously less than thenumber of immune-related genes of mammals, but the number of β-defensingenes and butyrophilin-like repertoires genes of the ducklings isobviously greater than that of the chickens. Since the poultry differsfrom the waterfowl in characteristics of immune response, the increasingspeed and the titer of antibodies of ducklings immunized with avianinfluenza inactivated vaccines are obviously lower than those of thechicken immunized with same. For example, after the chickens areimmunized with the avian influenza inactivated vaccine, the antibodytiter can reach 3 to 5 Log 2, 5 to 8 Log 2 and 8 to 10 Log 2respectively on 7th day, 14th days and 21st days, and the antibody titeris highest on 28th to 35th days; however, after the ducklings areimmunized with the avian influenza inactivated vaccine, the antibodytiter can reach 2 to 3 Log 2, 3 to 6 Log 2 and 5 to 8 Log 2 respectivelyon 7th day, 14th day and 21st day, and the highest antibody titer of 6to 9 Log 2 can be achieved on 28th to 35th days and can be maintainedfor 2 to 3 months.

Although there is a difference in the reports about the law of growthand decline for maternal antibodies of the ducklings, generally thetiter of the avian influenza maternal antibodies of the ducklings isdecreased from 8 to 10 Log 2 for the ducklings at the age of one day toa titer less than a critical point (4 to 6 Log 2) for the ducklings atthe age of 7 to 9 days, and to 0 to 2 Log 2 for the ducklings at the ageof 15 to 28 days. The maternal antibodies may interfere with the immuneeffect of the avian influenza inactivated vaccine on the ducklings andeven may produce immuno-suppression seriously. Since the breedingducklings generally have relatively high avian influenza antibodies, theproduced ducklings essentially have relatively high maternal antibodieswhich generally may protect the ducklings at the age of 1 to 15 days andmay help the ducklings to resist the infection of the high-pathogenicityavian influenza viruses. When the ducklings at the age of more daysreceive the avian influenza immunization, the immune dead time isexcessively long, which may increase the risk of the ducklings infectedwith the high-pathogenicity avian influenza viruses. Therefore, theducklings may be generally immunized with the avian influenzainactivated vaccine at the age about 10 days. Since the antibodies areslowly produced after the ducklings are immunized with the avianinfluenza inactivated vaccine, the ducklings immunized with the avianinfluenza vaccine have an immune dead time about two weeks. During thisperiod of time, the avian influenza valence is low, and the ducklingsare susceptible to the avian influenza infection, resulting in seriouseconomic loss. How to technically enable the ducklings immunized withthe avian influenza vaccine to quickly produce antibodies and maintainthe antibody level has an important significance on the duck farmingindustry.

SUMMARY

In order to overcome the defects in the prior art, an objective of thepresent invention is to provide a method for enabling ducklings toquickly produce anti-avian influenza antibodies and maintain antibodytiter.

In order to achieve the above-mentioned objective, the present inventionadopts a technical solution as follows:

a method for enabling ducklings to quickly produce anti-avian influenzaantibodies and maintain antibody titer includes: step 1) immunizingducklings at the age of 5 to 15 days: inoculating each duckling with anavian influenza inactivated antigen in an abdomen, and simultaneouslyintramuscularly or subcutaneously immunizing each duckling with the anavian influenza inactivated oil-emulsion vaccine.

Preferably, in step 1), the first immunization is performed on theducklings at the age of 8 to 10 days.

Preferably, in step 1), the dosage of the inactivated antigens is 0.1 to1 mL, and an HA valence is 7 to 12 Log 2.

Preferably, in step 1) the dosage of the inactivated antigens is to 0.3to 0.5 mL, and an HA valence is 9 to 10 Log 2.

Preferably, in step 1), the dosage of the inactivated oil-emulsionvaccines is 0.1 to 1 mL, and an HA valence is 7 to 12 Log 2.

Preferably, in step 1), the dosage of the inactivated oil-emulsionvaccines is 0.3 to 0.5 mL, and an HI valence is 9 to 10 Log 2.

Preferably, the method further includes a step 2): the duckling isre-immunized after 10 days from the first immunization: each duckling isinoculated with an avian influenza inactivated antigen in an abdomen.

Preferably, in step 2) the dosage of the inactivated antigens is 0.1 to1 mL, and an HA valence is 7 to 12 Log 2.

Preferably, in step 2), the dosage of the inactivated antigens is 0.3 to0.5 mL, and an HA valence is 9 to 10 Log 2.

Preferably, strains of the inactivated antigens include an H5 subtypeavian influenza strain and an H9 subtype avian influenza strain; and aproduction strain of the inactivated oil-emulsion vaccine includes an H5subtype avian influenza strain and an H9 subtype avian influenza strain.

Compared with the prior art, the present invention has the followingbeneficial effects:

1) through double effects of intraperitoneal inoculation with the avianinfluenza inactivated antigens and intramuscular or subcutaneousinjection and immunization with the avian influenza inactivatedoil-emulsion vaccines, the present invention enables ducklings toquickly produce the immune response, thereby quickly producinganti-avian influenza antibodies; and

2) the method for enabling ducklings to quickly produce the anti-avianinfluenza antibodies and maintain the antibody titer provided in thepresent invention is obviously higher in speed of producing the avianinfluenza antibodies than the traditional duck immunized with the avianinfluenza inactivated oil-emulsion vaccine, and the immune dead time ofthe duck immunized with the avian influenza inactivated vaccine iseffectively reduced by more than 7 days.

The present invention is further described below in detail incombination with specific embodiments.

DETAILED DESCRIPTION

The present invention provides a method for enabling ducklings toquickly produce anti-avian influenza antibodies and maintain antibodytiter, including: step 1) immunizing ducklings at the age of 5 to 15days: each duckling is inoculated with an avian influenza inactivatedantigen in an abdomen, and simultaneously intramuscularly orsubcutaneously immunizing the ducklings with avian influenza inactivatedoil-emulsion vaccines.

In the following specific embodiments, unless otherwise specified, theemployed reagents or test objects are commercially available.

In the following specific embodiments, the employed ducklings arecommercially-available local ducklings in Guangdong Province,inactivated antigens and inactivated oil-emulsion vaccines are fromGuangdong Wens Dahuanong Biotechnology Co. Ltd., and an inactivatedantigen strain includes an H5 subtype avian influenza strain and an H9subtype avian influenza strain, such as H5 Re-6 strain and H9 SS strain;and the inactivated oil-emulsion vaccine is H5+H9 subtype avianinfluenza inactivated vaccine, and the production strain thereofincludes an H5 subtype avian influenza strain and H9 subtype avianinfluenza strain, such as H5 Re-6 strain and H9 SS strain.

Embodiment 1

50 commercially-available newly hatched ducklings are obtained anddivided into six experimental groups, wherein group 1 is a control groupand is not immunized; group 2 receives first immunization at the age ofone day; group 3 receives the first immunization at the age of 5 days;group 4 receives the first immunization at the age of 8 days; group 5receives the first immunization at the age of 10 days; group 6 receivesthe first immunization at the age of 14 days; and groups 2 to 6 arere-immunized after 10 days from the first immunization.

For the ducklings in the experimental groups 1 to 6, blood is collectedfrom veins under duck wings on 0, 5th, 7th, 14th, 21st, 28th, 35th and42nd days after the first immunization, serum is isolated from theblood, and the valences (HI valences) of anti-avian influenza virusantibodies in the serum are uniformly determined Results are shown inTable 1.

In the present embodiment, the first immunization specifically includesthe following operation steps: each duckling is injected with 0.5 mL ofavian influenza inactivated antigen in the abdomen, and simultaneouslyis subcutaneously injected with 0.5 mL of avian influenza inactivatedoil-emulsion vaccine in the neck.

In the present embodiment the re-immunization treatment specificallyincludes the following operation step: each duckling is injected with0.5 mL of avian influenza inactivated antigen in the abdomen.

TABLE 1 Impact of Composite Immunization of Avian Influenza InactivatedAntigens and Inactivated Vaccines on Avian Influenza Antibodies ofDucklings Monitoring Time and Experi- Antibody Titer (HI Valence) mentalVac- 0 5th 7th 14th 21st 28th 35th 42nd group cine day day day day dayday day day Group 1 H5 6 5 4 2 0 0 0 0 H9 4 3 2 1 0 0 0 0 Group 2 H5 6 54 2 3 4 4 3 H9 4 3 4 3 4 4 4 3 Group 3 H5 2 5 5 6 6 7 5 4 H9 1 4 6 6 7 77 7 Group 4 H5 3 4 5 7 8 7 6 5 H9 2 6 7 8 9 9 8 8 Group 5 H5 2 5 6 7 8 88 7 H9 1 6 7 8 9 9 8 8 Group 6 H5 2 5 6 7 8 8 8 7 H9 1 6 8 8 9 9 9 8

The results show that after the ducklings are injected with the immuneavian influenza inactivated antigens, in the abdomens and subcutaneouslyinjected with the inactivated oil-emulsion vaccines, after 5 days, theH5 subtype avian influenza antibody titer can reach 4 to 5 Log 2 and theH9 subtype avian influenza antibody titer can reach 3 to 6 Log 2; forthe ducklings receiving the first immunization at the age of more than 5days, after 7 days, the H5 subtype avian influenza antibody titer canreach 5 to 7 Log, the H9 subtype avian influenza antibody titer canreach 6 to 8 Log 2, and the titer can be maintained for more than 35days; and for the ducklings receiving the first immunization at the ageof 8 to 10 days, the avian influenza H5 and H9 subtypes avian influenzaantibody titers are greater than 4 Log 2 after 5 days; after 7 days, theH5 subtype avian influenza antibody titer can reach 5 to 7 Log 2, the H9subtype avian influenza antibody titer can reach 7 to 9 Log 2, and thetiter can be maintained for more than 42 days.

Embodiment 2

50 commercially-available newly-hatched ducklings are obtained anddivided into 5 experimental groups, wherein group 1 is a control groupand is not immunized.

Groups 2 to 5 receive the first immunization at the age of 15 days,wherein each duckling in group 2 receives the first immunization bymeans of intramuscular injection with 0.5 mL of avian influenzainactivated antigen; each duckling in group 3 receives the firstimmunization by means of subcutaneous injection with 0.5 mL of avianinfluenza inactivated antigen in the neck; each duckling in group 4receives the first immunization by means of intranasal immunization with0.5 mL of avian influenza inactivated antigen; and each duckling ingroup 5 receives the first immunization by means of intraperitonealinjection with 0.5 mL of avian influenza inactivated antigen.

Groups 2 to 5 receive re-immunization after 10 days from the firstimmunization, and the re-immunization includes the following specificoperation steps:

Blood is collected from veins under wings of the ducklings on 0, 5th,7th, 14th, 21st, 28th, 35th and 42nd days after the first immunization,serum is isolated, the valences (HI valences) of anti-avian influenzavirus antibodies in the serum are uniformly determined; and results areshown in Table 2.

TABLE 2 Effects of Different Avian Influenza Inactivated AntigenImmunization Ways on Ducklings Monitoring Time and Antibody Experi-Titer (HI Valence) mental Vac- 0 5th 7th 14th 21st 28th 35th 42nd groupcine day day day day day day day day Group 1 H5 1 1 0 0 0 0 0 0 H9 2 1 00 0 0 0 0 Group 2 H5 1 2 3 1 0 0 0 0 H9 1.5 2 2 0 0 0 0 0 Group 3 H5 1 22 0 0 0 0 0 H9 2 3 3 2 1 0 0 0 Group 4 H5 1 3 2 1 0 0 0 0 H9 1 4 2 4 3 00 0 Group 5 H5 2 5 6.5 7 8 7 4 1 H9 1 6 7 8 9 8 5 2

It can be known from Table 2 that for the ducklings in group receivingthe immunization with the intraperitoneal injection way, after 5 daysfrom the first immunization, the H5 subtype avian influenza antibodytiter can reach as high as 5 Log 2, and the H9 antibody titer can reachas high as 6 Log 2 and both the H5 subtype avian influenza antibodytiter and the H9 subtype avian influenza antibody titer can be kept atmore than 7 Log 2 after 28 days from the first immunization. For theducklings receiving the immunization with the intramuscular injectionway, the neck subcutaneous injection way or the intranasal immunizationway, the H5 subtype avian influenza antibody titer and the H9 subtypeavian influenza antibody titer are very low.

Embodiment 3

50 commercially-available newly hatched ducklings are obtained anddivided into 5 experimental groups, wherein group 1 is served as acontrol group and is not immunized with the avian influenza inactivatedvaccine.

Groups 2 to 5 receive the first immunization at the age of 15 days,wherein each duckling in group 2 receives the intramuscular immunizationwith 0.1 mL of avian influenza inactivated antigen; each duckling ingroup 3 receives the intramuscular immunization with 0.3 mL of avianinfluenza inactivated antigen; each duckling in group 4 receives theintramuscular immunization with 0.5 mL of avian influenza inactivatedantigen; and each duckling in group 5 receives the intramuscularimmunization with of avian influenza inactivated antigen. There-immunization is performed after 10 days from the first immunization,and the immunization dosage and the immunization way of there-immunization are the same as those of the first immunization.

Blood is collected from veins under wings of the ducklings on 0, 5th,7th, 14th, 21st, 28th, 35th and 42nd days after the first immunization,serum is isolated, the valences (HI valences) of anti-avian influenzavirus antibodies in the serum are uniformly determined; and results areshown in Table 3.

TABLE 3 Effects of Intramuscular Immunization with Avian InfluenzaInactivated Antigen on Ducklings Monitoring Time and Antibody Experi-Titer (HI Valence) mental Anti- 0 5th 7th 14th 21st 28th 35th 42nd groupgen day day day day day day day day Group 1 H5 2 1 0.5 0 0 0 0 0 H9 3 21 0 0 0 0 0 Group 2 H5 1 5 6 6 5 3 2 0 H9 1.5 6 7 7 5.5 3 2 0 Group 3 H51 6 7 7 5.5 4 2 1 H9 2 7 8 9 6 4 3 1 Group 4 H5 2 6 8 8 7 5 3 1 H9 1 7 89 7 6 4 2 Group 5 H5 2 6 8 9 7 6 3 1 H9 1 7 8 9 8 7 4 2

It can be known from Table 3 that when the ducklings receive theintraperitoneal immunization with 0.1 to 1 mL of avian influenzainactivated antigen, the avian influenza H5 subtype and H9 subtype avianinfluenza antibody titers in a duckling body can reach more than 5 Log 2after 5 days from the immunization; and when the ducklings receive theintraperitoneal immunization with 0.3 to 0.5 mL of avian influenzainactivated antigen, the avian influenza H5 subtype and H9 subtype avianinfluenza antibody titers in the duckling body, can be kept at 5 Log 2for more than 21 days.

Embodiment 4

50 commercially-available newly hatched ducklings are obtained anddivided into 5 experimental groups, wherein group 1 is served as acontrol group and is not immunized with the avian influenza inactivatedvaccine.

Ducklings in groups 2 to 5 receive the first immunization by means ofsubcutaneous injection with the avian influenza inactivated vaccine atthe age of 15 days; each duckling in group 2 is immunized with 0.1 mL ofavian influenza inactivated vaccine; each duckling in group 3 isimmunized with 0.3 mL of avian influenza inactivated vaccine; eachduckling in group 4 is immunized with 0.5 mL of avian influenzainactivated vaccine; and each duckling in group 5 is immunized with 1 mLof avian influenza inactivated vaccine.

Blood is collected from veins under wings of the ducklings on 0, 5th,7th, 14th, 21st, 28th, 35th and 42nd days after the first immunization,serum is isolated, the valences (HI valences) of anti-avian influenzavirus antibodies in the serum are uniformly determined; and results areshown in Table 4.

TABLE 4 Effects for Immunizing Ducklings with Avian InfluenzaInactivated Vaccine Monitoring Time and Antibody Experi- Titer (HIValence) mental Vac- 0 5th 7th 14th 21st 28th 35th 42nd group cine dayday day day day day day day Group 1 H5 2 2 1 1 0 0 0 0 H9 1 1 1 0 0 0 00 Group 2 H5 1 1 2 2 3 4 5 5 H9 1.5 2 3 4 5 6 6 6 Group 3 H5 1 2 3 4 5 66 6 H9 1.5 2 4 7 8.5 8 8.5 8 Group 4 H5 2 1 2 3 5 6 7 6 H9 1 1 3 6 8 8.59 9 Group 5 H5 0.5 1 2 3 5 6 6 6 H9 0.5 2 5 6 8 9 9 8

It can be known from Table 4 that after 21 days from that the ducklingsare immunized with the avian influenza inactivated vaccine, the H5subtype avian influenza specific antibody-valence (HI valence) is 3 to 5Log 2 and the H9 subtype avian influenza specific antibody valence (HIvalence) is 5 to 8 Log 2; and the avian influenza specific antibodyvalences are relatively low;after 0 to 14 days from the immunization andinsufficient to play a role in protection.

The above embodiments are only preferred embodiments of the presentinvention and may not be used to limit the protection scope of thepresent invention. Any insubstantial variations and replacements made bythose skilled in the art based on the present invention shall fallwithin the protection scope claimed by the present invention.

We claim:
 1. A method for enabling ducklings to quickly produceanti-avian influenza antibodies and maintain antibody titer, comprising:step 1) performing first immunization on ducklings at the age of 5 to 15days: inoculating each duckling with an avian influenza inactivatedantigen in an abdomen, and simultaneously intramuscularly orsubcutaneously immunizing each duckling with an avian influenzainactivated oil-emulsion vaccine.
 2. The method according to claim 1,wherein in step 1), the first immunization is performed on the ducklingsat the age of 8 to 10 days.
 3. The method according to claim 1, wherein,in step 1), the dosage of the inactivated antigens is 0.1 to 1 mL, andan HA valence is 7 to 12 Log
 2. 4. The method according to claim 1,wherein, in step 1), the dosage of the inactivated antigens is 0.3 to0.5 mL, and the HA valence is 9 to 10 Log
 2. 5. The method according toclaim 1, wherein, in step 1), the dosage of the inactivated oil-emulsionvaccines is 0.1 to 1 mL, and an HI valence is 7 to 12 Log
 2. 6. Themethod according to claim 1, wherein, in step 1), the dosage of theinactivated oil-emulsion vaccines is 0.3 to 0.5 mL, and the HI valenceis 9 to 10 Log
 2. 7. The method according to claim 1, further comprisingstep 2) re-immunizing the ducklings after 10 days from the firstimmunization: inoculating each duckling with the avian influenzainactivated antigen in an abdomen.
 8. The method according to claim 7,wherein in step 2), the dosage of the inactivated antigens is 0.1 to 1mL, and the HA valence is 7 to 12 Log
 2. 9. The method according toclaim 7, wherein, in step 2), the dosage of the inactivated antigens is0.3 to 0.5 mL, and the HA valence is 9 to 10 Log
 2. 10. The methodaccording to claim 1, wherein in the step 1), strains of the inactivatedantigens comprise an H5 subtype avian influenza strain and an H9 subtypeavian influenza strain; and a production strain of the inactivatedoil-emulsion vaccine comprises an H5 subtype avian influenza strain andan H9 subtype avian influenza strain.